Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels

Recombinant TWIK2 channels produce weak basal background K+ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this variability is due to a unique cellular trafficking. We identified three different sequence signals responsible for the preferential expression of TWIK2 in the Lamp1-positive lysosomal compartment. Sequential inactivation of tyrosine-based (Y(308)ASIP) and di-leucine-like (E266LILL and D(282)EDDQVDIL) trafficking motifs progressively abolishes the targeting of TWIK2 to lysosomes, and promotes its functional relocation at the plasma membrane. In addition, TWIK2 contains two N-glycosylation sites (N(79)AS and N(85)AS) on its luminal side, and glycosylation is necessary for expression in lysosomes. As shown by electrophysiology and electron microscopy, TWIK2 produces functional background K+ currents in the endolysosomes, and its expression affects the number and mean size of the lysosomes. These results show that TWIK2 is expressed in lysosomes, further expanding the registry of ion channels expressed in these organelles

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions

Étude des mécanismes d'adressage dans la voie de sécrétion régulée du précurseur de la neurotensine

L'adressage des précurseurs hormonaux vers la voie de sécrétion régulée des cellules neuroendocrines est un mécanisme mal connu et très controversé. Les travaux réalisés au cours de cette thèse ont eu pour objectif de caractériser dans la pro-neurotensine/neuromedine N, un précurseur de neuropeptides, les motifs nécessaires à la sécrétion régulée des produits de maturation. Par mutagenèse dirigée et transfection dans des cellules pourvues de la voie de sécrétion régulée, nous avons caractérisé une répétition de motifs discrets indispensable à l'adressage régulé. Cette séquence est capable d'entraîner la sécrétion régulée d'une protéine constitutive. La présence de ce motif ne modifie pas les propriétés d'agrégation in vitro. Le signal caractérisé est impliqué dans l'adressage et la maturation du précurseur. Les résultats de stimulation obtenus dans des cellules incapables de le maturer révèlent une sécrétion régulée même sans clivage. Des larges formes incomplètement maturées issues du précurseur peuvent donc être sécrétées. Ces produits existent dans des situations physiologiques et pathologiques. L'analyse de leurs propriétés biologiques révèlent qu'elles sont capables de se lier au récepteur des peptides et d'induire un signal intracellulaire. Elles représentent donc des ligands potentiels avec des propriétés spécifiques, dont une résistance accrue à la dégradation.NICE-BU Sciences (060882101) / SudocSudocFranceF

The family of K 2P channels: salient structural and functional properties

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Silent but not dumb: how cellular trafficking and pore gating modulate expression of TWIK1 and THIK2

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Silencing of the Tandem Pore Domain Halothane-inhibited K + Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane

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The intracellular Na(+)/H(+) exchanger NHE7 effects a Na(+)-coupled, but not K(+)-coupled proton-loading mechanism in endocytosis

SummaryVesicular H+-ATPases and ClC-chloride transporters are described to acidify intracellular compartments, which also express the highly conserved Na+/H+ exchangers NHE6, NHE7, and NHE9. Mutations of these exchangers cause autism-spectrum disorders and neurodegeneration. NHE6, NHE7, and NHE9 are hypothesized to exchange cytosolic K+ for H+ and alkalinize vesicles, but this notion has remained untested in K+ because their intracellular localization prevents functional measurements. Using proton-killing techniques, we selected a cell line that expresses wild-type NHE7 at the plasma membrane, enabling measurement of the exchanger’s transport parameters. We found that NHE7 transports Li+ and Na+, but not K+, is nonreversible in physiological conditions and is constitutively activated by cytosolic H+. Therefore, NHE7 acts as a proton-loading transporter rather than a proton leak. NHE7 mediates an acidification of intracellular vesicles that is additive to that of V-ATPases and that accelerates endocytosis. This study reveals an unexpected function for vesicular Na+/H+ exchangers and provides clues for understanding NHE-linked neurological disorders

TWIK1, a unique background channel with variable ion selectivity.

International audienceTWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells

Does sumoylation control K2P1/TWIK1 background K+ channels?

A novel model for the regulation of cell excitability has recently been proposed. It originates from the observation that the background K(+) channel K2P1 (TWIK1) may be silenced by sumoylation in Xenopus oocytes and that inactivation of the putative sumoylation site (mutation K274E) gives rise to robust current expression in transfected COS-7 cells. Here, we show that only the mutation K274E, and not K274R, is associated with an increase of K2P1 current density, suggesting a charge effect of K274E. Furthermore, we failed to observe any band shift by western blot analysis that would confirm an eventual sumoylation of K2P1 in COS-7 cells and oocytes

Predicting hERG repolarization power at 37°C from recordings at room temperature

International audienceDear Editor, Loss-of-function and gain-of-function mutations in the KCNH2 gene cause long and short-QT syndromes (LQTS or SQTS), respectively, predisposing to life-threatening F I G U R E 1 Repolarization power of wild-type (WT) hERG as a function of temperature. A simplified and optimized action potential was applied (AP-clamp) by an automated patch clamp system in 384-well plates on HEK293 cells stably expressing hERG. (A) Mean (± SEM) current recordings during AP-clamp at various temperatures (in pA, n = 194, 211, 114 and 172 cells at 22-37 • C, respectively). Dashed line: AP time course (voltage scale: right Y axis). (B) Mean (± SEM) current recordings during AP-clamp at 27 • C for AP of various durations (see inset for APs; for currents: n = 168−254 for Time x 0.5 to x 5). The small inward current observed in (A) and (B), when the AP is returning to resting values, is attributed to contamination of the intracellular solution by the extracellular Tyrode solution, intrinsic to the cell catch process in the automated patch clamp (see supplementary information). (C) Mean (± SEM) time integral of the recorded currents: repolarization power, at various temperatures versus time factor (n = 155−232, 168−254, 79−133 and 144−173 at 22-37 • C, respectively). Horizontal dashed line: repolarization power at 37 • C: 22.3 pA.s. (D) As in (B), at 27 • C, after time and current corrections using various factors from 1.5 to 3 on the respective recordings. For example, for the recordings obtained during the AP of 2 x duration, the time was divided by 2 and the current multiplied by 2. Note the overlap of the current corrected by the factor of 2 at 27 • C (black) with the reference current obtained during the standard AP at 37 • C (red)